The envelope glycoprotein (env) of human immunodeficiency virus (HIV) plays a central role in the pathogenicity of AIDS but the arrangement of subunits in this protein is still unknown. Scanning transmission electron microscopy (STEM) has been applied to determine the subunit arrangement of purified, cloned derivatives of the envelope protein gp140 in both HIV and SIV. Mass measurements were also made on virion-derived SIV and HIV env protein, obtained by large-scale virus isolation and cross-linking prior to detergent extraction. Macromolecular complexes were adsorbed onto thin carbon supports and were plunge-frozen, freeze-dried and imaged in dark-field STEM at low electron dose. Images were analyzed quantitatively using a calibration standard of tobacco mosaic virus to determine the distributions of molecular weights and hence the oligomeric states of the proteins. From the mass measurements, it was found that the complexes of virion-derived SIV and HIV env as well as the SIV gp140 were composed of trimers. This finding was further supported by the observation of triangular or tri-lobed structures in electron micrographs. The results were also confirmed by sedimentation data obtained from analytical ultracentrifugation. Mass measurements of HIV gp140 assemblies showed a more heterogeneous population of oligomers with evidence of dimers and tetramers in addition to trimers. Our results indicate that SIV env maintains a trimeric conformation before and after fusion activation and further suggest that SIV gp140 may be a good model for env presented on the virion surface in future immunological and structural studies.